R-DeeP: Proteome-wide and Quantitative Identification of RNA-Dependent Proteins by Density Gradient Ultracentrifugation

Mol Cell. 2019 Jul 11;75(1):184-199.e10. doi: 10.1016/j.molcel.2019.04.018. Epub 2019 May 7.


The comprehensive but specific identification of RNA-binding proteins as well as the discovery of RNA-associated protein functions remain major challenges in RNA biology. Here we adapt the concept of RNA dependence, defining a protein as RNA dependent when its interactome depends on RNA. We converted this concept into a proteome-wide, unbiased, and enrichment-free screen called R-DeeP (RNA-dependent proteins), based on density gradient ultracentrifugation. Quantitative mass spectrometry identified 1,784 RNA-dependent proteins, including 537 lacking known links to RNA. Exploiting the quantitative nature of R-DeeP, proteins were classified as not, partially, or completely RNA dependent. R-DeeP identified the transcription factor CTCF as completely RNA dependent, and we uncovered that RNA is required for the CTCF-chromatin association. Additionally, R-DeeP allows reconstruction of protein complexes based on co-segregation. The whole dataset is available at http://R-DeeP.dkfz.de, providing proteome-wide, specific, and quantitative identification of proteins with RNA-dependent interactions and aiming at future functional discovery of RNA-protein complexes.

Keywords: CTCF; R-DeeP; RNA; RNA dependence; RNA-binding protein; RNase; density gradient; mass spectrometry; proteome-wide; proteomics.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Centrifugation, Density Gradient / instrumentation
  • Centrifugation, Density Gradient / methods*
  • Chromatin / chemistry
  • Chromatin / metabolism
  • Gene Expression Regulation
  • Gene Ontology
  • HeLa Cells
  • Humans
  • Information Dissemination
  • Internet
  • Molecular Sequence Annotation
  • Protein Binding
  • Protein Interaction Maps*
  • Proteome / classification
  • Proteome / genetics*
  • Proteome / metabolism
  • Proteomics / methods
  • RNA / genetics*
  • RNA / metabolism
  • RNA-Binding Proteins / classification
  • RNA-Binding Proteins / genetics*
  • RNA-Binding Proteins / metabolism
  • Transcription Factors / classification
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism


  • Chromatin
  • Proteome
  • RNA-Binding Proteins
  • Transcription Factors
  • RNA